| Tauroursodeoxycholic acid(TUDCA)is considered as the peculiar and the main active ingredient of Bear Gall Powder.Based on the state project of “Major New Drugs Innovation and Development” for key technology and preclinical research for breeding the bear bile powder in vitro(2014ZX09301306-007),the chitosan microspheres activated by glutaraldehyde were used as the carrier to immobilize the critical enzyme of 7?-hydroxysteroid dehydrogenase(7?-HSDH)and 7?-hydroxysteroid dehydrogenase(7?-HSDH)in previous by our research group,which was preformed to satisfy the requirement of industrial production.And 7?-HSDH and 7?-HSDH could be utilized to catalyze taurochenodeoxycholic acid(TCDCA)to TUDCA in vitro.However,the chitosan microspheres were split easily due to their poor mechanical strength during the continuous reaction operation.Therefore,the selection of a new carrier with excellent properties and high mechanical strength was a key point of the present study.Epoxy resin,with high mechanical strength and good physical and chemical properties,is widely used as a carrier of the immobilized enzyme.However,a low activity was obtained of the enzyme immobilized by epoxy resin in general.Therefore,a bifunctional group of amino-epoxy resin was obtained by chemically amino-modification of epoxy resin,followed by the immobilization of 7?-HSDH and 7?-HSDH which was conducted with amino-epoxy resin as the carrier in further research,and the transformation of TCDCA with the co-immobilized enzymes was also studied.Research showed that amino-epoxy resin obtained by EDA modification is a suitable carrier with excellent mechanical strength in the immobilization of 7?-HSDH and 7?-HSDH.The properties of immobilized enzyme(high activity,operation stability and storage stability)are finally contributed to the industrial production.The main research content and results are listed as follows:(1)The preparation and characterization of the bifunctional amino-epoxy resin.The bifunctional amino-epoxy resin was obtained through the method of modifying amino group by EDA to epoxy resin,and the results of infrared spectroscopy indicated that the second funcational amino group was successfully introduced.There is no diffidence of the surface on the carrier was observed by the SEM.It is obviously from the conduction of amino-group density by the titration that the modification reaction reached equilibrium after 6 hours.The surface on the carrier is positively charged in the pH 2~10;The contact angle was reduced from 51.5° to 33.4° after modification,which is the avidence of higher hydrophilic property.(2)The establishment of enzyme-immobilized process.The enzyme activities could be obtained with the highest value,when 7?-HSDH and 7?-HSDH were immobilized on the amino-epoxy resin with the amino modified density of 67.6 ?mol/g.Under the same immobilized conditions,the enzyme activity and the enzyme loading rate were significantly improved when 7?-HSDH and 7?-HSDH immobilized on the amino-epoxy resin(amino modified density was 67.6 ?mol/g)compared with the ones immobilized on epoxy resin,and the activity of enzyme was increased by 5.8 and 1.25 times respectively.Different immobilization principles of the two carriers resulted in differences in enzyme loading rate and enzyme activity.The conditions for the immobilization of amino-epoxy resin were optimized from the former conclusion.The optimum immobilization time of 7?-HSDH and 7?-HSDH were tested,which was proved to be 4 h and 3 h,respectively,and the optimal immobilization temperature of both was 15 °C,the optimal immobilization pH value was 7.5.Under this condition,the highest enzyme activity of immobilized enzyme can be obtained.(3)The study on the enzymatic properties of immobilized enzyme.The optimum reaction temperature for immobilized 7?-HSDH and 7?-HSDH was 20 °C and 25 °C,respectively.The optimum pH for immobilized 7?-HSDH and 7?-HSDH was 10 and 5.5,respectively.Furthermore,comparing the Km value before and after immobilization,the affinity of 7?-HSDH to TCDCA and NADP+ was decreased and the affinity of 7?-HSDH to T-7-KLCA was increased,however,the affinity to NADPH was decreased.Compared with free enzymes,the thermal stability of immobilized enzymes were both improved obviously.At 35 °C,the residue activity of the free 7?-HSDH and 7?-HSDH was 50.67% and 58% respectively,while the higher enzyme activity was achieved on immobilized enzyme with 73.52% and 84.08% respectively.It was worthy to mentioned that the residue activity of immobilized 7?-HSDH and 7?-HSDH could be respectively remained 79.4% and 83.1% after 6 times reaction,and residue activity of the immobilized 7?-HSDH and 7?-HSDH still remained 49.1% and 66.4% after 4 weeks in the condition of pH 8.0,4 °C.The former figure was the evidence proved the well operation stability and storage stability of immobilized enzymes.(4)The study on co-immobilization of 7?-HSDH and 7?-HSDH.At the catalytic condition of pH 8.0 and 25 °C,the yield of TUDCA can be reached 31.57% when 7?-HSDH was immobilized before 7?-HSDH.The ratio of 7?-HSDH to 7?-HSDH was 3:1,a high yield(34.3%)of TUDCA was obtained.In the combined enzymatic catalyzed reaction system,the highest yield of TUDCA canbe obtained when the concentration of TCDCA was 6 mM,however,substrate inhibition was found above 6 mM.The combination of bi-enzyme was used to achieve the recycling of coenzyme NADP+,and the conversion of TCDCA to TUDCA was dominant when the concentration of NADP+ was 2 mM. |
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